Random priming advice needed

afc at gnv.ifas.ufl.edu afc at gnv.ifas.ufl.edu
Tue Dec 1 08:42:10 EST 1992


In article <1149.2b1a6f73 at nphi.fi>, tronni at nphi.fi writes:
> Hi all bio-people in the net!
> 
> I would like to hear opinions concerning the efficiency
> of "random priming" systems for labeling linear DNA.
<deleted>

We have been using the BRL rp kit for the last year or so.  Before
that we were using the BRL nick translation kit.

We get essentially complete incorportation of 32P label into DNA.
If you calculate it out, this represents 1:1 copying of the input
DNA.  The specific activity is about 25x higher than nick translation.

However, our hybridizations don't seem to work any better.  The 
problem seems to be that the BRL kit has way too much primer, and
that incorporation is into little tiny stuff that doesn't hybridize.
We have recently cut the amount of primer that we add by a factor of
12.  Incorporation is not lowered and it seems to hybridize better.
The lesson is, specific activity is *not* what you should be trying
to optimize.  Length is also critical (for probes at least).

Andrew Cockburn
USDA

> Cheers,
> 
> Tapani Ronni
> National Public Health Institute
> Mannerheimintie 166
> 00300 Helsinki
> FINLAND



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